Med23 deletion exhibited aggravated carbon tetrachloride (CCl4)-induced liver fibrosis, with enhanced chemokine production and inflammatory infiltration as well as increased hepatocyte regeneration. Mechanistically, the orphan nuclear receptor RAR-related orphan receptor alpha (RORα) activates the expression of the liver fibrosis-related chemokines C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10), which is suppressed by the Mediator subunit MED23. We further found that the inhibition of Ccl5 and Cxcl10 expression by MED23 likely occurs because of G9a -mediated H3K9 dimethylation of the target promoters. Collectively, these findings reveal hepatic MED23 as a key modulator of chemokine production and inflammatory responses and define the MED23-CCL5/CXCL10 axis as a potential target for clinical intervention in liver fibrosis.Liver fibrosis, often associated with cirrhosis and hepatocellular carcinomas, is characterized by hepatic damage, an inflammatory response, and hepatic stellate cell (HSC) activation, although the underlying mechanisms are largely unknown. Here, we show that the transcriptional Mediator complex subunit 23 (MED23) participates in the development of experimental liver fibrosis. Compared with their control littermates, mice with hepatic  Liver fibrosis is characterized by hepatic damage, an inflammatory response, and hepatic stellate cell activation, but the underlying mechanisms remain unclear. This study reveals that the transcriptional Mediator subunit MED23 regulates the pathogenesis of liver fibrosis by controlling the production of inflammatory cytokines such as CCL5 and CXCL10. Liver fibrosis is a major chronic liver disease that can progress to more severe liver cirrhosis and eventually cause hepatocellular carcinoma, accompanied by significant mortality , 2. SuffSeveral studies to date have demonstrated that hepatic inflammation plays an important role in the underlying pathogenesis of liver fibrosis, which subsequently leads to the recruitment and activation of hepatic stellate cells (HSCs) as well as the excess production of extracellular matrix (ECM) proteins , 5, 6. IMed23 (med23Δli) do not display abnormal hepatic histology and function, Med23 ablation improves hepatic glucose metabolism, as reflected by enhanced glucose tolerance and insulin sensitivity. Notably, liver-specific Med23 deletion prevented high-fat diet (HFD)- and genetic-induced obesity and reduced the related pathological consequences of this condition ) mice fed with an HFD, inflammatory response was significantly reduced [Ccl5 and Cxcl10 expression positively in hepatocytes, thereby aggravating the immune response in liver. These observations provide a promising strategy for inhibiting the activation of inflammatory responses by RORα antagonists in liver disease development. Besides, it is worthwhile to note that MED23 may regulate Ccl5 and Cxcl10 expression via alternative molecules. For example, C/EBPβ, which determines differential gene activation by interacting with the Mediator MED23 subunit [Ccl5 and Cxcl10 expression. However, we observed that knockdown of C/EBPβ only slightly affected the increase in Ccl5 and Cxcl10 expression caused by Med23 silencing in AML12 cells, suggesting that C/EBPβ may not be the major player in this model system.To better understand how hepatocyte MED23 regulates the expression of elopment , 35 and elopment , 22, as elopment , 36. Howelopment . RORα neelopment . In vitrelopment . By contelopment . Additio subunit , was alsWe observed that MED23 suppresses the transcriptional activity of RORα in hepatocytes through G9a-mediated H3K9 dimethylation . In a prMed23 deletion, we further observed increased compensatory hepatocyte proliferation in med23Δli mice compared with that in control mice. These data suggest that Med23-deficient hepatocytes display increased regenerative capability after CCl4 injury, although the detailed mechanisms remain to be further investigated. Perhaps the cross talk between hepatocytes and nonparenchymal liver cells, such as HSCs and immune cells, contributes to the induction of hepatocyte proliferation; and Med23 deficiency in hepatocytes results in increased immune cell infiltration and HSC activation while providing additional paracrine signals  to stimulate hepatic proliferation. Conceivably, modulating paracrine signaling may present interesting approaches for liver regeneration or liver cancer treatment.After chronic liver injury, surviving liver parenchyma cells expand to replenish the surrounding dead cells to maintain homeostasis . HoweverIn conclusion, we demonstrate for the first time that the Mediator subunit MED23 plays an important role in experimental liver fibrosis and provide new insight into the molecular mechanisms of inflammatory responses that initiate fibrotic changes upon liver injury. In the future, targeting the MED23/chemokine axis may provide intervention strategies for liver fibrosis.All animal experiments were conducted using mice bred at and maintained in our animal facility according to the guidelines of the Institutional Animal Care and Use Committee of the Shanghai Institute of Biochemistry and Cell Biology (IACUC-PR01).med23f/f mice were generated via homologous recombination [Albumin-cre mice were a generous gift from Dr. Lijian Hui . To generate hepatocyte-specific med23Δli mice, med23f/f mice were crossbred with Albumin-cre mice as described previously [The bination . Albumineviously . All ani4 (1:4 vol/vol in sunflower oil from Sigma) every 3 days for 4 weeks to induce liver fibrosis. All mice were euthanized 72 hours after the final CCl4 injection, and livers were collected for histological, cytological, biochemical, and molecular analyses. To induce acute liver injury, 8-week-old male mice were injected with CCl4 as described above; all animals were euthanized 24 hours after CCl4 injection.The 8-week-old male mice were intraperitoneally injected with 0.6 μl/g body weight CCl2.The AML12 cell line was kindly provided by Dr. Jianguo Song   three times. After immersion in diluted normal goat serum, sections were sequentially incubated with anti-mouse F4/80 antigen PE  overnight at 4°C. Nuclei were stained with DAPI for 5 minutes after two washes with PBS.TUNEL assays were performed using an Apoptosis DNA Fragmentation Assay Kit  to detect apoptotic cells. All images were visualized using a U-RFL-T microscope . The positive cells or areas were counted or measured in at least five fields on each slide using ImageJ (NIH) software.4 injection; and after an additional 24 hours, the mice were euthanized, and F4/80 immunofluorescence staining was performed to detect the efficiency of macrophage elimination.A single dose of control liposomes or clodronate liposomes (10 μl/g body weight) was intravenously injected into the tail vein of 8-week-old male mice to deplete macrophages. After 24 hours, the mice were administered a CClThe purity and integrity of the extracted RNA were confirmed using an Agilent Bioanalyzer. Libraries were prepared from 100 ng of total RNA , and pair-ended sequencing was performed on an Illumina HiSeq 2500 using bar-coded multiplexing and a 150-bp read length, yielding a median of 34.1 M reads per sample. After quality assessment of the corresponding FASTQ files with FastQC, adapter sequences were trimmed from the reads. Read alignment and junction finding were accomplished using TopHat, and differential gene expression was analyzed with Cuffdiff using the University of California Santa Cruz (UCSC) mm10 assembly as the reference sequence. In addition, genes with an expression fold change of >|2| were selected for further analysis (accession code GSE137457).Med23 knockdown was established according to the manufacturer’s recommendation (Clontech). Briefly, retroviruses were generated following the cotransfection of recombinant pSiren-RetroQ plasmids with the pCL10A1 helper plasmid into 293T cells using Lipofectamine 2000 . Culture supernatants containing retroviruses were harvested 48 hours after transfection and filtered through 0.22-μm filters. Virus-containing supernatants were mixed with 4 μg/ml Polybrene  and 10 μM HEPES  and were then added to the plated cells for spin infection . Cells were selected with puromycin (Sigma-Aldrich) 24 hours after spin infection. The targeting sequences are listed in An AML12 cell line with stable Med23 knockdown or the control AML12 cells were seeded into 6-well plates . The next day, these cells were transfected with the control siRNA or RORα siRNA (10 μM) using RNAiMax transfection reagent. At 48 hours after transfection, cells were collected for protein and RNA extraction. The targeting sequences are listed in AML12 cells were transfected with the indicated siRNAs (10 μM) using RNAiMax transfection reagent  according to the manufacturer’s instructions. At 48 hours after transfection, cells were collected for protein and RNA extraction. For g, 4°C, 5 minutes), the small pieces were cross-linked with a final concentration of 1% formaldehyde in PBS on a shaker for 20 minutes at room temperature and neutralized by the addition of glycine to a final concentration of 0.125 M for 5 minutes. After washing with precooled PBS, the small pieces were homogenized in ChIP lysis buffer , and the pellet was collected, followed by sonication. The following procedures were performed as described previously [Mouse livers (0.2 g) for ChIP assays were freshly isolated and cut into small pieces in precooled PBS supplemented with protein inhibitors. After centrifugation (450eviously . The ant5 cells per well overnight. These cells were then transfected with a luciferase reporter plasmid and pRL-TK (Promega) along with various expression constructs, as indicated, by Lipofectamine 2000 . All wells were supplemented with control empty expression vector plasmids to keep the total amount of DNA constant. The cells were harvested and subjected to dual-luciferase reporter assays after 24–36 hours of transfection according to the manufacturer’s protocol (Promega).The dual-luciferase reporter assay was performed as described . Brieflyt test or Mann-Whitney test was used to determine significant differences between data sets using GraphPad Prism (version 5.0). Differences were considered statistically significant when P ≤ 0.05.All data are presented as the means ± SEM. A two-tailed unpaired Student S1 Figmed23f/f and med23Δli mice fed with a MCD diet . (B) Liver sections from med23f/f and med23Δli mice fed with a MCD diet were stained with HE and α-SMA, and representative views are shown. (C) Quantification of the α-SMA-positive area in livers of med23f/f and med23Δli mice . (D) The total protein was extracted from whole livers of med23f/f and med23Δli mice fed with a MCD diet and analyzed by western blotting using the indicated antibodies. HSP70 was used as a loading control. (E) Quantification of the α-SMA and Col1a1 levels in livers of med23f/f and med23Δli mice  and normalized to the HSP70. Error bars denote SEM from three independent experiments . Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. For underlying data, see med23, Mediator complex subunit 23; med23Δli, liver-specific knockout of Med23; med23f/f, med23-floxed; qRT-PCR, quantitative real-time PCR.(A) qRT-PCR analysis of fibrosis-related factors in whole-liver extracts of (TIF)Click here for additional data file.S2 Figmed23f/f and med23Δli mice (n = 6–7 per group). (B) Analysis of liver weight, body weight, and liver/body weight in med23f/f and med23Δli mice (n = 7 per group). Data are presented as means ± SEM. *P < 0.05, **P < 0.01. For underlying data, see 4, carbon tetrachloride; med23, Mediator complex subunit 23; med23Δli, liver-specific knockout of Med23; med23f/f, med23-floxed.(A) Serum ALT, AST, and albumin were measured in (TIF)Click here for additional data file.S3 Figmed23f/f and med23Δli mice after acute CCl4 administration for 24 hours. (B) Serum ALT and AST were measured in med23f/f and med23Δli mice after acute CCl4 administration for 24 hours . (C) Representative images of α-SMA and F4/80 staining in the liver sections of med23f/f and med23Δli mice after acute CCl4 administration for 24 hours. The liver paraffin sections were stained with α-SMA antibody, and liver frozen sections were stained with F4/80 antibody (red). DAPI (blue) was used for nuclear counterstaining. (D) Quantification of F4/80-positive cells from the liver sections of med23f/f and med23Δli mice . (E-F) qRT-PCR analysis of Acta2 (E) and F4/80 (F) expression in whole-liver extracts of med23f/f and med23Δli mice. The mRNA expression was normalized to Gapdh . (G) Western blotting analysis of total protein extracted from whole livers of med23f/f and med23Δli mice using the indicated antibodies. GAPDH was used as a loading control. (H) qRT-PCR analysis of fibrosis-associated genes expression in whole-liver extracts of med23f/f and med23Δli mice . Data are presented as means ± SEM. **P < 0.01. For underlying data, see 4, carbon tetrachloride; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HE, hematoxylin–eosin; med23, Mediator complex subunit 23; med23Δli, liver-specific knockout of Med23; med23f/f, med23-floxed; qRT-PCR, quantitative real-time PCR.(A) Representative views of HE staining in the liver sections of (TIF)Click here for additional data file.S4 Fign = 19; cirrhosis, n = 58; HCC, n = 38). (B-C) qRT-PCR analysis of RORα (B) and Ccl2 (C) expression in AML12 cells after RORα overexpression. The mRNA expression was normalized to Gapdh (n = 4 per group). (D) qRT-PCR analysis of Ccl2 expression in shCtrl and shMed23 AML12 cells after RORα knockdown. The expression was normalized to Gapdh (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For underlying data, see Gapdh, glyceraldehyde 3-phosphate dehydrogenase; HCC, hepatocellular carcinoma; MED23, Mediator complex subunit 23; qRT-PCR, quantitative real-time PCR; RORα, RAR-related orphan receptor alpha; shCtrl, negative control vector containing scrambled shRNA; shMed23, shRNA against Med23.(A) The published database (GSE14323)  was util(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data4, carbon tetrachloride; FC, fold change; med23, Mediator complex subunit 23; med23Δli, liver-specific knockout of Med23; RNA-seq, RNA sequencing.CCl(XLS)Click here for additional data file.S3 DataMed23, Mediator complex subunit 23.IPA, Ingenuity Pathway Analysis; (XLS)Click here for additional data file.S1 TableshRNA, short hairpin RNA.(DOCX)Click here for additional data file.S2 TablesiRNA, small interfering RNA.(DOCX)Click here for additional data file.S3 TableChIP-qPCR, chromatin immunoprecipitation followed by quantitative PCR.(DOCX)Click here for additional data file.S4 TableqRT-PCR, quantitative real-time PCR.(DOCX)Click here for additional data file.S1 Text(PDF)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file. 7 Jun 2019Dear Dr WANG, Thank you for submitting your manuscript entitled "The Mediator subunit MED23 regulates inflammatory responses and liver fibrosis" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.Please re-submit your manuscript within two working days, ie. by Jun 09 2019 11:59PM.https://www.editorialmanager.com/pbiologyLogin to Editorial Manager here: http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit. During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review. plosbiology@plos.org if you have any queries relating to your submission.Feel free to email us at Kind regards,Di JiangPLOS Biology 17 Jul 2019Dear Dr WANG,Thank you very much for submitting your manuscript "The Mediator subunit MED23 regulates inflammatory responses and liver fibrosis" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by three independent reviewers.In light of the reviews (below), we will not be able to accept the current version of the manuscript, but we would welcome resubmission of a revised version that takes into account the reviewers' comments. Our Academic Editor advises that you should include another injury model, primary hepatocyte data and data to show that this is relevant to human liver disease. It is not necessary to provide evidence that this is pathway is dysregulated in human disease, but you are encouraged to provide it if it is already available as it will increase the impact of the study. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.Your revisions should address the specific points made by each reviewer. Please submit a file detailing your responses to the editorial requests and a point-by-point response to all of the reviewers' comments that indicates the changes you have made to the manuscript. In addition to a clean copy of the manuscript, please upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type. You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.http://journals.plos.org/plosbiology/s/file?id=9411/plos-biology-formatting-checklist.pdf. It is helpful if you format your revision according to our requirements - should your paper subsequently be accepted, this will save time at the acceptance stage.Before you revise your manuscript, please review the following PLOS policy and formatting requirements checklist PDF: http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information . For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.Please note that as a condition of publication PLOS' data policy  to discuss this if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not wish to submit a revision and instead wish to pursue publication elsewhere, so that we may end consideration of the manuscript at PLOS Biology.We expect to receive your revised manuscript within two months. Please email us . While this data is relevant, most of this data is descriptive and may be related to the same mechanisms, meaning that it is likely looking at the same observation from different angles.2. The paper relies on a single model and the rationale for studying MED23 is not well explained. The authors should employ more liver injury models to exclude that their findings are specific to CCl4, a standardized model for liver fibrosis research but without much human relevance. Moreover, they should analyze MED23 in patients. It is not clear at all whether MED23 activity is altered in human fibrosis and contributes to disease progression.3. The authors do not sufficiently investigate whether MED23 may simply regulate responses to CCl4 and liver injury. The authors show ALT levels in the chronic CCl4 model, where liver injury is typically low towards the end of experiments and where differences may be difficult to reveal. There is no data on serum ALT in the acute injury models and not investigation of key factors that regulate CCl4 metabolization and liver injury such as Cyp expression/activity. Moreover, the assessment of apoptosis by TUNEL assay and cleaved caspase is not highly relevant as most of CCl4-induced cell death is necrosis. In light of these, it would also have been advantageous to use additional models that do not rely on biactivation of a toxin and/or mostly necrotic cell death (see comments above).4. The clodronate and in vitro experiments do not conclusively prove a role for MED23 via the regulation of hepatocyte Ccl5 and Cxcl10. The authors should have analyzed this in primary hepatocytes and possbily used single cell RNA-seq to compare Ccl5 and Cxcl10 expression between MED23 and floxed controls as well as between hepatocytes and other cell types in the MED23-deleted mice - this way they could have strengthened their hypothesis that hepatocytes represent the main Ccl5- and Cxcl10-secreting cell type in mice with MED23 in hepatocytes. Finally, it would have been important to show the role of Ccl5 and/or Cxcl10 by deleting these in hepatocytes in vivo - which can nowadays be achieved by hepatocyte-specific silencing or deletion methods without lengthy breedings.5. The authors found that Hgf, was higher in MED23-deleted livers than in control livers. It is not clear why this is the case as HGF is normally expressed by stellate cells and endothelial cells. As Hgf is an important modulator of liver injury and regeneration, this could also represent a major mechanism by which MED23 regulates responses to liver injury, rather than the postulated Ccl5 and Cxcl10 pathway in hepatocytes.Minor points1. The authors state “the expression of Desmin, another marker of HSCs activation, was also higher in med23Δli mouse livers than in med23f/f mouse livers”. Desmin is not a marker of activation - it is a marker of HSC and an increase may reflect an increased number of HSC in injured livers.Reviewer #2: Liver fibrosis is a wound-healing response that is a consequence of chronic liver diseases. Despite the widespread of this disease, the underlying molecular mechanisms remain largely unknown. Development of novel effective therapies for liver fibrosis requires identifying effective molecular targets. The focus of this research is the study of the molecular mechanism underlying liver fibrosis that is a relevant and important issue.The manuscript “The Mediator subunit MED23 regulates inflammatory responses and liver fibrosis” is dedicated to studying of the role MED23 subunit of the Mediator complex in the development of experimental liver fibrosis. The authors showed that mice with hepatic Med23 deletion exhibited aggravated CCl₄-induced liver fibrosis. The manuscript provides evidence that MED23 negatively regulates the expression of the hepatic chemokines CCL5 and CXCL10 via suppressing the activity of the retinoid-related orphan nuclear receptor, RORα.In summary, this manuscript is of value for the further development of our knowledge of the molecular mechanism underlying liver fibrosis and will help the development of more effective treatment options. I would recommend the manuscript for publication, but I have the following comments.CRITIQUEMy main problem with this manuscript is that the authors made several deficiencies, which cast doubt on the conclusions and correctness of the model proposed.1. Necessary to give data about the number of mice in all experiments.2. Fig 6F. Necessary to show Western-Blot for proving the effectivity of the knocked down Rora in protein level.3. Fig 6I. Necessary to describe this experiment with all details in Materials and Methods. I have doubts about the conclusions made by the authors. Controls have significant differences. It should be discussed in the manuscript with an explanation. In the current moment, conclusion about a functional interaction between RORα and MED23 in controlling Ccl5 and Cxcl10 expression needs additional experiments.4. Fig 6J. The dual luciferase reporter assay should be described in Materials and Methods.5. Fig 6K. This experiment should be described with details in the manuscript.6. Fig 7A. Necessary to show a base level of signal for ChIP on non-promoter site of the genome.7. I would like to ask the authors to provide more discussion in the text. I highly recommend expanding the Introduction part and give more information about the factors used in the work.8. In this manuscript, a significant part of the data is the results of the qRT-PCR. I would like to ask the authors to pay more intention for presenting this type of data. Nowadays good practice to use two or three genes for normalization especially if the differences between experiment and control less than 2-folds.Reviewer #3: The authors study the role of Med23 in experimental models of liver fibrosis (carbon tetrachloride) in conditional knock out mice using Albumin-CRE, Med23 flox/flox mice. The authors find that deletion of Med23 in hepatocytes and cholangiocytes leads to increased liver fibrosis, decreased apoptosis but increased hepatocyte proliferative activity and increased inflammatory activity, though the characterization of inflammatory cell composition remains a bit superficial. In well designed in vitro studies using murine hepatocyte cell line AML12 and other models, the authors show that Med23 cooperates with ROR� in negatively regulating cytokines CCL5 and CXCL10. The authors show that Med23 deletion causes decreased H3K9me2 occupancy among the promoter of CCL5 and CXLC10 thus identifying novel mechanism for Med23. Though the authors ultimately do not show the biological relevance of Med23-CCL5/CXLC10 axis in liver fibrosis and inflammation e.g. by showing abrogation of increased liver fibrosis in double knock out mice of both Med23 and CCL5/CXCL10 or data on Med23 expression levels in human liver datasets from public databases , the shown data are of very good quality and experiments are well designed. The conclusions are sound and the manuscript well written with the topic within the journals scope, I thus recommend the acceptance of the manuscript for publication.Minor comments: additional information regarding the catalog numbers of siRNA / plasmids used to knock down targets would be informative for readers interested in replicating the studies. 20 Sep 2019Attachmentrebuttal letter.docxSubmitted filename: Click here for additional data file. 29 Oct 2019Dear Dr WANG,Thank you for submitting your revised Research Article entitled "Mediator MED23 regulates inflammatory responses and liver fibrosis" for publication in PLOS Biology. I have now obtained advice from two of the original reviewers and have discussed their comments with the Academic Editor. The reviews are included below. We recognise the concerns that continue to be raised by reviewer 1, but after discussing them with the academic editor, we think that these can be satisfactorily addressed by ensuring that these potential limitations are clearly flagged for the reader. We will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by reviewer 1. Please also make sure to address the data and other policy-related requests noted at the end of this email.We expect to receive your revised manuscript within two weeks. Your revisions should address the specific points made by reviewer 1. In addition to the remaining revisions and before we will be able to formally accept your manuscript and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. 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We ask that you notify us as soon as possible if you or your institution is planning to press release the article.https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript. To submit your revision, please go to Please do not hesitate to contact me should you have any questions.Sincerely,Di JiangPLOS Biology------------------------------------------------------------------------ETHICS STATEMENT:Please add an Ethics Statement subsection at the beginning of the Methods section. The Ethics Statements in the submission form and Methods section of your manuscript should match verbatim. ------------------------------------------------------------------------DATA POLICY:Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.------------------------------------------------------------------------Reviewer remarks:Reviewer #1: While the authors did additional work including a second model, most of my concerns remain.1. The second model (MCD model) was not included in the actual paper and mechanisms seem to be different from the CCl4 model and not be mediated by the proposed inflammatory mechanism. Moreover, the MCD model is nowadays viewed as inadequate model of NASH fibrosis and other dietary models are preferred (but it is adequate for purely mechanistic studies on fibrosis)2. The authors have not convinced this reviewer that the main role of MED23 is not related to protection from injury. This appears to be THE driver of the phenotype - everything else is likely secondary unless proven otherwise by functional experiments . 3. This reviewer is not convinced that hepatocyte-secreted CCl5 and CXCL10 are the main drivers of fibrogenesis in this model. First of all, hepatocytes are not thought to be the main source of these chemokines in the liver. Second of all, hepatocyte isolations from CCl4-treated livers are inherently contaminated with inflammatory cells. The suggestion of using single cell RNA-seq as a platform that can avoid these problems was not taken up by the authors. In the absence of single cell RNA-seq analysis, the authors would need to provide evicence for this unlikely hypothesis, e.g. by hepatocyte-specific silencing of CCl5 and CXCL10 .Reviewer #2: The manuscript “Mediator MED23 regulates inflammatory responses and liver fibrosis” was modified, taking into account all the comments. The authors have conducted additional experiments to cement their findings further, as suggested by editors and reviewers. The text of the manuscript was corrected and significantly improved. I recommend this manuscript to be accepted for publication in its current form. 14 Nov 2019AttachmentThe new response .docxSubmitted filename: Click here for additional data file. 15 Nov 2019Dear Dr WANG,On behalf of my colleagues and the Academic Editor, Luke A Noon, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology. The files will now enter our production system. 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The final article will be published to the same URL, and all versions of the paper will be accessible to readers.PRESS biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact me if I can provide any assistance during the production process.Kind regards,Hannah HarwoodPublication Assistant, PLOS Biologyon behalf ofDi Jiang,Associate EditorPLOS Biology


Identification of social risk factors and the promotion of stress coping mechanisms and mental resilience are topics of interest in the field of mental health. The relationships between risk- or tolerability-associated factors and task-related hemodynamic responses in the prefrontal cortex (PFC) in adolescents may have important implications for mental health challenges. The purpose of this study was to investigate the relationship between task-related PFC hemodynamic activities and subjective well-being or lifestyle habits using wearable near-infrared spectroscopy (NIRS). In this study, after sample refinement to reduce heterogeneity, 20 university students were included in verbal working memory (VWM) task analyses and 21 were included in spatial working memory (SWM) task analyses. The task-related hemodynamic responses were detected using wearable NIRS. To assess the risk- or tolerability-associated factors, the levels of positive and negative affect were assessed using the Subjective Well-Being Inventory (SUBI) and lifestyle habits (such as gaming) were evaluated using a nine-item questionnaire. There was a positive correlation between SUBI positive affect and VWM task-related oxy-hemoglobin signal changes in the right dorsolateral PFC (DLPFC), underlining the significance of subjective well-being as an important independent emotional domain and suggesting the possibility of the differential objective evaluations of subjective well-being in the right PFC. Negative correlations between PFC activities during both VWM and SWM tasks at the left DLPFC and the number of game playing days in 1 week were also statistically significant, suggesting the presence of modality-non-specific hemodynamic regulation by habitual game playing. Each correlation was still robust after the elimination of major confounding impacts. Although further replication studies are warranted to confirm these preliminary results, this investigation of the relationship between task-related PFC hemodynamic activities and emotional domains or lifestyle habits might have clinical significance with regard to primary prevention of mental health issues in university students. To our knowledge, this is the first demonstration of these relationships with the use of wearable NIRS, which enables measurement under near natural conditions and is easy to use in schools or workplaces. Identification of modifiable social risk factors across the lifespan and the promotion of prevention are topics of interest in the field of global mental health  is a recurrent site of integration between human affect and cognition, and significant complex interactions between PFC functions and human affect have been demonstrated in neuroimaging studies . Written informed consent was obtained from all participants after the purpose, procedure, risks, benefits, personal information management, and voluntary nature of participation had been explained to them. In the analysis processes, all data were anonymized and the researchers were blinded to the links between the data and personal information.A total of 28 participants were initially enrolled. The participants included in the final analysis were selected using the criteria described below . The recruitment was sequential and the participants were all Japanese-speaking and undergraduate or graduate students at Kagoshima University. Handedness was assessed using the Edinburgh Handedness Inventory . The working memory tasks, VWM and SWM, were controlled by a laptop computer that was connected and synchronized with the other laptop for NIRS analyses. In each trial of both tasks, the target stimulus (S1) was visualized for 1.5 s, and following a 7.0 s delay, the probe (query) stimulus (S2) was exhibited for 2.0 s or until the response submission . The signal-averaging of the task-related NIRS responses was validated as a biomarker of PFC working memory functions, and the tasks used in this study were essentially the same as those in previous NIRS studies  that can be identified by the individual syllable, the type change between S1 and S2 compels the participant to retain the verbal (letter) information of S1 for the response to the S2 query in the VWM task. Four Hiragana characters were presented as S1, and a Katakana character was presented as S2 . The parThe procedure for this experiment is illustrated in via the local wireless network. In the probe unit, the emitters and detectors are alternately arranged in two rows at an inter-probe distance of 3 cm  was used to measure PFC activity. The attached probe unit (a wearable package as a headset), composed of eight emitters and eight detectors, can fully cover the participant’s forehead . The det of 3 cm . Because of 3 cm . The emi of 3 cm , using l of 3 cm .A plug-in based application  developed by Hitachi ARL on MATLAB  was used to analyze the average task-related oxy-Hb signal changes across participants  was used to confirm the difference in oxy-Hb signal change averages between students with the habit and those without the habit. The correlations among questionnaire scores, task performance (reaction time and accuracy), and age and the task-related oxy-Hb signal change were evaluated. In box-and-whisker plotting for the task-related oxy-Hb signal changes, cases whose values were outside the box edges by more than 1.5 times the interquartile range were objectively determined as outliers. The inter-relationships between questionnaire scores and age were investigated in the final group of valid participants. To evaluate sex differences in the task-related oxy-Hb signal change and questionnaire scores, the average across participants was compared between males and females for each task by Student’s t-test and Wilcoxon rank sum test. For the false discovery rate (FDR) correction for multiple comparisons among a total of 18 channels, the FDR threshold was set to 0.05 . The noise-free and baseline-corrected data sequence were also utilized to evaluate the task-related oxy-Hb signal change. In each trial block of each participant, the PFC activation value was calculated , between game playing time in 1 day (h/day) and in 1 week , between days of drinking and days of gaming in 1 week , and between the hours of smartphone use and hours of internet use in 1 day . Although there were significant differences between sexes in the average smartphone use (h) per day  and the number of days of exercise in 1 week , no significant differences were observed between sexes in the other variables .For the final group of 21 valid participants , we calculated the mean and SD of the SUBI and lifestyle questionnaire scores. In the SUBI, the mean scores were 41.19 ± 5.99 for positive affect and 50.19 ± 4.71 for negative affect. In the lifestyle questionnaire, the mean scores were 0.79 ± 0.75 h/day and 3.33 ± 3.04 days/week for playing games, 1.14 ± 1.77 days/week for drinking, 3.10 ± 3.22 h/day for internet use, 2.62 ± 0.86 h/day for smartphone use, and 0.95 ± 1.63 h/week for physical exercise. Correlations between the SUBI and lifestyle questionnaire scores and age are presented in t = 2.57–7.72, p ≤ 0.0094, q < 0.05; t = 2.04–6.84, p ≤ 0.0280, q < 0.05; Signal-averaging of the task-related oxygenation process at each channel across all analyzed participants provided the grand average waveforms for each task . The delThe positions of channels that recorded task-related oxy-Hb signal changes that were significantly correlated with questionnaire scores are indicated in the channel array figure , 8A, 9A.r = 0.61, p = 0.0046, q < 0.05; channel 8: r = 0.62, p = 0.0031, q < 0.05; r = 0.68, p = 0.0013; channel 8: r = 0.70, p = 0.0008). A significant negative correlation between oxy-Hb signal changes during the VWM task and the number of game playing days in 1 week was revealed in channel 16, at the left DLPFC . Student’s t-test confirmed a significant difference in oxy-Hb signal change averages between students with game habits (n = 14) and those without game habits . The oxy-Hb signal changes during the VWM task did not significantly correlate with the SUBI negative affect score (r = −0.24 to 0.45), age (r = −0.18 to 0.36), number of drinking days in 1 week (r = −0.61 to −0.04), average internet use hours per day (r = −0.27 to 0.45), average smartphone use hours per day (r = −0.27 to 0.5), average game playing hours per day (r = −0.47 to 0.04), or number of days of exercise in 1 week (r = −0.35 to 0.44). In addition, there was no significant correlation between the VWM task-related PFC activation and task performance , and there was no significant difference between sexes in the VWM-task related oxy-Hb signal changes (t = −2.1 to 2.2).For the VWM task, significant positive correlations between the task-related oxy-Hb signal changes and SUBI positive affect scores were observed in two channels (channels 7 and 8) at the right DLPFC (channel 7: r = −0.33 to 0.49) or SUBI negative affect scores (r = −0.51 to 0.60). A significant negative correlation between oxy-Hb signal changes during the SWM task and the average number of game playing days in 1 week was detected in channel 16, at the left DLPFC  was observed in the same channel as that in the VWM task (n = 20). A Student’s t-test confirmed a significant difference in oxy-Hb signal change averages between students with game habits (n = 14) and those without game habits . Significant negative correlations were also obtained between SWM task-related oxy-Hb signal changes and the number of drinking days in 1 week in channels 12 and 17, at the frontal pole (FP) and left DLPFC, respectively . Student’s t-tests confirmed differences in oxy-Hb signal change averages between students with drinking habits (n = 9) and those without drinking habits (n = 12) in channels 12  and 17 . On the other hand, in the relationship between drinking habits and PFC functions, the exclusion of participants without drinking habits  may have affected the correlation analysis. However, the negative correlation at channel 12 was not statistically significant in nine participants who had drinking habits , and the negative correlation at channel 17 disappeared with removal of non-drinkers . Additional outlier removal did not make the relationship significant at channel 17 . The oxy-Hb signal changes during the SWM task did not significantly correlate with age (r = −0.18 to 0.36), the average internet use hours per day (r = −0.50 to 0.11), the average smartphone use hours per day (r = −0.56 to 0.36), the average game playing hours per day (r = −0.61 to −0.05), or the number of days of exercise in 1 week (r = −0.18 to 0.35). In addition, there was no significant correlation between the SWM task-related PFC activation and task performance , and there was no significant difference between sexes in the SWM-task related oxy-Hb signal changes (t = −0.41 to 2.46).The oxy-Hb signal changes during the SWM task did not significantly correlate with the SUBI positive affect scores . The significant negative correlation between oxy-Hb signal changes during both working memory tasks and the number of game playing days in 1 week was also robust at channel 16, even after the elimination of the effects of the other variables . However, the significant correlation between SWM task-related PFC activation and the number of drinking days in 1 week disappeared at both channel 12 and 17 after the elimination of the effects of the other variables .Three variables  that exhibited significant correlations with task-related oxy-Hb signal changes might influence each other with regard to their significance. In particular, there was a statistically significant correlation between gaming habits and drinking habits . To evalIn the framework of the National Institute of Mental Health’s research domain criteria (RDoC), environmental factors provide the broad context and the RDoC matrix consists of the functional domains and their units of analysis  emotion vs. non-semantic (right) emotion  were not evaluated in our investigation. Further analyses with larger sample sizes and more detailed considerations of various confounding factors are warranted to ensure the significance and implications of our results, particularly those associated with individual well-being. In order to investigate the contribution of trait variables, developmental trends, twin-studies, and consideration of psychopathology or personality variables may be important. The study of patients with mental illness may be required to investigate the influence of subjective ill-being.In this study of university students with relatively similar backgrounds, we demonstrated that PFC hemodynamic activities during working memory tasks might be modifiable by positive affect (as assessed by the SUBI) and lifestyle habits, which may impact mental health. Although further replication studies are warranted to confirm these preliminary results, subsequent and more detailed investigations of the relationships between task-related hemodynamic activities and emotional domains  or lifestyle habits may contribute to the elucidation of the neural mechanism concerning vulnerability and resilience to mental illness and neural processes of disease manifestation. Because the wearable NIRS device can provide a quiet, non-invasive, and low-constraint experimental environment, and does not require any additional equipment or a large space, it is easy to use in schools or workplaces, where primary prevention is an important topic of interest.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.This study was approved by the Institutional Review Board Ethics Committee of the Health Service Center, Kagoshima University (accession no.: H26-4-1). Written informed consent was obtained from all participants after the purpose, procedure, risks, benefits, personal information management, and voluntary nature of participation had been explained to them. In the analysis processes, all data were anonymized and the researchers were blinded to the links between the data and personal information.YK designed and performed the experiment, analyzed the data, and wrote the manuscript. JN administered the SUBI and lifestyle questionnaire, and contributed to NIRS measurement. TF created the figures and performed analyses to double-check the results. CK provided technical advice regarding statistical analyses. MN performed Wilcoxon rank sum test and contributed to the manuscript revision. AS contributed to the manuscript revision. All authors have read and approved this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium berghei ANKA were classified into two groups, namely, the ALI/ARDS and non-ALI/ARDS groups, according to lung histopathology. The lungs of uninfected mice were used as a control group. The location and expression of SP-D in the lung tissues were investigated by immunohistochemical staining and Western blot analysis. In addition, the level of SP-D in plasma and lung homogenate was measured by an enzyme-linked immunosorbent assay. Immunohistochemical staining of SP-D was significantly increased in the lung tissues of the malaria-infected mice in the ALI/ARDS group compared with that in the malaria-infected mice in the non-ALI/ARDS group and the mice in the control group (p < 0.05). The levels of SP-D in the plasma and lung homogenate were significantly increased in the malaria-infected mice in the ALI/ARDS group compared with those in the malaria-infected mice in the non-ALI/ARDS group and the mice in the control group (p < 0.05). There was a significant positive correlation between SP-D in the plasma and SP-D in the lung homogenate . In conclusion, this study demonstrated increased expression levels of SP-D in the lung tissue and high levels of plasma SP-D in the malaria-infected mice with ALI/ARDS compared with those in the mice in the other groups. The current study supports that the elevation of the plasma SP-D level may provide useful biological confirmation of the diagnosis of ALI/ARDS during malaria infection.Surfactant protein D (SP-D) is in the collectin family of C-type lectins and plays an important role in the regulation of inflammation and the innate immune defense against pathogens. This protein has been proposed as a biomarker for acute lung injury. However, the expression of SP-D in the lung and the circulating levels of SP-D during malaria infection have received limited attention. Therefore, the aim of this study was to determine the location and expression of the SP-D protein in lung tissue and to measure the plasma level of SP-D in experimental malaria-associated acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Malaria-infected mice induced by Malaria-associated acute lung injury/malaria-associated acute respiratory distress syndrome (ALI/ARDS) is prevalent in malaria-endemic areas, with a mortality rate of approximately 80% . PreviouSP-D is a collagenous calcium-dependent lectin that is involved in surfactant homeostasis and the innate immune defense within the lung –19. This6 Plasmodium berghei ANKA-infected red blood cells to induce ALI/ARDS according to methods described in previous studies [ P. berghei, Strain ANKA, MRA-311, contributed by Thomas F. McCutchan. The control mice received saline solution via intraperitoneal injection. After infection, parasitemia was determined daily by microscopic analysis of thin blood smears from tail blood after Giemsa staining. All the infected mice with signs of ALI/ARDS and the control mice were sacrificed at day 13 after infection. Blood was collected by cardiac puncture, and plasma was prepared and stored at -20°C until it was used for SP-D measurements. The lung tissues were removed immediately for immunohistochemistry and Western blot analyses. The lung tissues of the malaria-infected mice were categorized into two groups (ALI/ARDS and non-ALI/ARDS groups) in accordance with the evaluation of lung histopathology by an expert pathologist. Specifically, the lungs with ALI/ARDS were diagnosed based on the observation of pulmonary edema, alveolar hemorrhage, alveolar septal thickness, and hyaline membrane formation. Only 50% (n = 5 mice out of 10) of the P. berghei-infected mice developed ALI/ARDS.All animal procedures and protocols were approved by the Animal Ethics Committee from Walailak University (protocol no. 003/2018). All experiments were performed in accordance with relevant guidelines and regulations. Six- to ten-week-old male DBA/2 mice were purchased from BioLASCO Co., Ltd. . The mice were maintained in the Animal Experiment Building at Walailak University and housed in a conventional animal house with free access to food and fresh water. The animal house was set at 22 to 24°C with a standard 12 hr light/dark cycle. The mice were intraperitoneally injected with 1 × 10 studies , 24. TheSections of formalin-fixed and paraffin-embedded lung tissues were incubated at 60°C for 1 hr followed by deparaffinization and rehydration. After rehydration, endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. Subsequently, the lung sections were incubated with citrate buffer (pH 6.0) for 30 min  in a microwave to improve antigen retrieval. After they were washed with Tris-buffered saline (TBS), the sections were incubated in normal goat serum for 30 min at room temperature to prevent nonspecific binding. Then, the sections from control, ALI/ARDS, and non-ALI/ARDS groups were incubated with a rabbit polyclonal primary antibody against SP-D  for 1 hr at room temperature. Next, the sections were incubated in a diluted biotinylated secondary antibody for 30 min followed by incubation for 30 min with VECTASTAIN ABC Reagent . Visualization was achieved with diaminobenzidine (DAB)  incubation for 5 min. The sections were counterstained with Mayer's hematoxylin and examined under a light microscope. The primary antibodies were omitted from the negative control sections, which were incubated with either TBS or normal goat serum. For semiquantitative analysis of SP-D-positive staining, each immunostained section was randomly assessed for staining intensity in 10 microscopic fields at a high magnification (400X). The SP-D staining intensity was scored as follows: 0 = negative staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining, as previously described . Finallyμl of a biotinylated antibody was added to each well and incubated at 37°C for 60 min. After washing of the well plate with phosphate-buffered saline (PBS), avidin biotin peroxidase was added to each well and incubated for 30 min at 37°C. After a further washing step, tetramethylbenzidine (TMB) solution was added to the well plate as a substrate and incubated for 15-20 min at 37°C in the dark. Finally, the optical density in each well was measured at a wavelength of 450 nm in a microplate reader within 30 min after adding the stop solution. All the assays were performed in duplicate.The levels of SP-D in the plasma and lung tissue were measured using a mouse SP-D ELISA kit according to the manufacturer's protocol . Briefly, each plasma and protein sample was added to a 96-well plate and incubated for 90 min at 37°C. After discarding the contents in the wells without washing, 100 μg) from each sample was resolved by reducing and separating by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE)  at room temperature (RT). Then, the samples were transferred onto an activated polyvinylidene fluoride (PVDF) membrane . The membrane was blocked with 5% nonfat milk  in TBS-0.01% Tween 20 (TBS-T) for 2 hr at RT. The membrane was further incubated overnight at 4°C with a rabbit polyclonal antibody against SP-D  as the primary antibody and a rabbit polyclonal antibody against β-actin  as the positive control. After being washed with TBS-T, the membrane was incubated with HRP-conjugated goat anti-rabbit secondary antibodies  for 1 hr at RT. Finally, the protein bands on the membrane were detected using a chemiluminescent substrate (ECL Plus) kit . The molecular weights of the detected protein bands were estimated using standard proteins  ranging from 10 to 250 kDa. Quantitative densitometric analysis of the gel bands was conducted using ImageJ software , and the signal intensity of each band was normalized using the corresponding β-actin loading control. All the assays were performed in duplicate.For Western blots, lung tissues were homogenized in cell lysis buffer  containing phenylmethylsulfonyl fluoride , which is a protease inhibitor, to prepare the protein samples. The total protein concentration of the lung tissues was determined using a Bradford Protein Assay Kit  with bovine serum albumin (BSA) as the standard. Total protein  was used for statistical analyses. Data in this study was not normally distributed determined by the Kolmogorov–Smirnov test. Mann–Whitney p < 0.05) (n = 5 mice per group).The results of the immunohistochemical studies demonstrated that the cytoplasmic positive staining for SP-D was located in the alveolar epithelial cells, especially alveolar type II cells and alveolar macrophages . The lunp < 0.05)  .p = 0.009). In addition, the mean levels of SP-D in the lung homogenates were significantly elevated in the malaria-infected mice with ALI/ARDS (200.50 ± 17.15 ng/mL) compared with those in the malaria-infected mice in the non-ALI/ARDS group (87.71 ± 9.95 ng/mL) and the mice in the control group (84.07 ± 7.14 ng/mL) (p = 0.011). Spearman's rank correlation analysis revealed that there was a significant positive correlation between SP-D in the plasma and SP-D in the lung homogenate . Moreover, there was a significant positive correlation between the levels of SP-D in the plasma and the staining intensity score for SP-D  (The levels of SP-D in the plasma and lung homogenates were measured by ELISA . The mea= 0.005) . Plasmodium berghei ANKA as an animal model of malaria associated with ALI/ARDS. The mice were classified as having ALI/ARDS based on the presence of pulmonary edema, alveolar hemorrhage, alveolar septal thickness, and hyaline membrane formation. These pathological findings corresponded to the histopathology of human ALI/ARDS reported by Punsawad et al. [th day after infection was 22.46% and 29.42% in the non-ALI/ARDS and ALI/ARDS groups, respectively (p = 0.008).In our study, we used DBA/2 mice infected withd et al. . The mea Plasmodium falciparum [ in vitro and in vivo studies are needed to further understand the role of SP-D in alveolar macrophages during ALI/ARDS.In the present study, we investigated the expression and circulation of the SP-D protein in a mouse model of malaria associated with ALI/ARDS. Our study demonstrated that positive immunoreactivity for SP-D was localized to the alveolar type II cells and alveolar macrophages. These findings are consistent with previous studies, which reported the location of SP-D positive staining in alveolar type II cells and alveolar macrophages , 20, 21.lciparum  and Hz [lciparum  contribulciparum , 16. Ourlciparum . High exlciparum , 20. How Pseudomonas aeruginosa-induced pneumonia and sepsis model in mice, compared to wild-type and humanized SP-D transgenic mice, mice lacking pulmonary SP-D are more susceptible to bacterial infection and exhibit more severe lung injury associated with bacterial pneumonia [α (TNF-α) and a 2-fold increase in the number of pulmonary monocytes/macrophages have been observed in Sftpd−/− mice compared with those in wild-type mice [In the present study, an increased level of plasma SP-D was observed in the malaria-infected mice with ALI/ARDS compared with that in the malaria-infected mice in the non-ALI/ARDS group and the mice in the control groups. This finding is consistent with previous studies that showed increased serum SP-D levels in an experimental model of ALI , 9, 10. neumonia . In accoype mice . In humaype mice , 12, 14 ype mice . In contype mice . The curype mice , (ii) enype mice , 21. FurSome limitations of the present study should be addressed. We did not perform double staining with primary antibodies against the alveolar type II cells and lung macrophage markers. We did not determine the time courses of the BALF and plasma levels of SP-D in the malaria-infected mice with ALI/ARDS. An increased level of SP-D in BALF has been reported in a rat model of a bleomycin-induced lung injury , 9. It hThe current study found increased expression levels of SP-D in lung tissues and high levels of plasma SP-D in malaria-infected mice with ALI/ARDS compared with those in the mice in the other groups. This study supports that elevated levels of plasma SP-D may be a useful biological confirmation of the presence of ALI/ARDS during malaria infection. Further investigations are needed for time course measurements of plasma SP-D and for the correlation of SP-D levels with clinical data in malaria patients with ALI/ARDS.


BP tracking coefficients were calculated from Spearman's rank-order correlations. Predictive values and logistic regression models were used to forecast t1-BP above the hypertension threshold from t0-BP as well as from baseline and follow-up hypertension risk factors. BP tracking was moderate (0.33-0.50 for SBP and 0.19-0.39 for DBP) with no statistically significant differences between sex and age groups. Baseline hypertensive BP was the strongest independent predictor of hypertensive BP at follow-up (OR 4.3 and 3.4 for age groups 3-10 and 11-17 years) after adjusting for sex, BMI trajectories, birthweight, parental hypertension, and age-group dependent-sports/physical activity. However, the positive predictive value of baseline hypertensive BP for hypertensive BP at follow-up in 3- to 10-year olds was only 39% (34% in 11- to 17-year olds) and increased only moderately in the presence of additional risk factors. Our analysis with population-based data from Germany shows that BP in children and adolescents tracks only moderately over six years. BP in childhood is the strongest independent predictor of future BP but its predictive value is limited.Blood pressure (BP) tracking from childhood to adulthood has two aspects: the ranking stability relative to others over time and the prediction of future values. This study investigates BP tracking in children and adolescents in Germany in the context of hypertension risk factors. BP was measured and analyzed in 2542 participants of the German Health Examination Survey for Children and Adolescents (t Blood pressure (BP) elevation is much more common among children and adolescents than previously thought. Moreover, BP in childhood correlates with BP in adulthood ; hence cBP tracking correlation has two aspects: the stability of BP ranking relative to others over a specified period of time  and the predictability of future values from baseline values. Meta-analyses have reported moderate tracking coefficients of 0.42-0.38 for SBP and 0.41-0.28 for DBP from childhood to adulthood .Therefore, the aim of this study is twofold: (1) to investigate six year BP tracking correlation coefficients from childhood to adolescence/young adulthood in a large population-based sample with a broad baseline age range of 3 to 17 years and to illustrate whether this aspect of tracking is stronger in subgroups with additional hypertension risk factors; and (2) to analyse the predictability of future BP values above the hypertension threshold from previous BP and hypertension risk factors and to quantify this predictability using different measures of association including odds ratios (OR) and predictive values.0 2003-2006; participants aged 3 to 17 years) and the MoMo follow-up  with complete BP data sets and who have not taken antihypertensive medication at t0 (unfortunately we do not have information on medication use at t1). The proportion of complete data sets was higher for those 3-10 years old at t0 than for those 11-17 years old. Analyses were stratified by age group: 3-10 years at t0 and 9-17 years at t1 vs. 11-17 years at t0 and 18-24 years at t1. We therefore excluded 144 participants who did not fall into these two groups in order to have a clear cutoff in the age group analysis and not mix both hypertension definitions , leaving 2542 participants  for analysis. Additional follow-up data were available from the telephone interview KiGGS1 2009-2012 [The German Health Interview and Examination Survey for Children and Adolescents (KiGGS) is a nationwide study based on a stratified population-registry sample of 17,641 participants aged 0–17 years living in Germany (response rate 67%), including 14,835 participants aged 3-17 years [0) and in the MoMo study 2009-2012 (t1). At both time-points, BP measurements were taken in the sitting position on a height-adjustable chair with a backrest, the right forearm resting on a table at the level of the heart, the elbow slightly bent, the legs uncrossed, and the feet placed firmly on the floor. Four cuff sizes  were available, which had to cover at least two-thirds of the upper arm length . Two readings of systolic (SBP), diastolic blood pressure (DBP), mean arterial BP, and heart rate were obtained at a two-minute interval with an automated upper arm oscillometric device  after a nonstrenuous part of the examination and an additional 5-minute rest in both surveys [BP measurement methods were standardized and followed the same protocol in KiGGS0 2003-2006  according to a standardized protocol [2 (in m2) and rounded to 3 digits. BMI was categorized as underweight, normal weight, overweight, and obese according to the International Obesity Task Force (IOTF) cutoffs defined by the age-specific percentiles in <18-year olds, which at the age 18 and older correspond to the adult cutoff points for overweight 25 kg/m2 and obesity 30 kg/m2 [0 and t1 in 4 categories: “persistently low or normal” defined as under- or normal weight at t0 and at t1; “resolution” as overweight or obese at t0 and normal or underweight at t1; “persistently overweight/obesity” (at t0 and at t1); and “incident overweight/obesity” as under- or normal weight at t0 and overweight or obese at t1.In addition, weight and height were measured at tprotocol . BMI was30 kg/m2 . BMI tra0: in children aged 3 to 10 years it was assessed through parent-reported frequency of sports activities per week  [Information on physical activity was collected only in tan once)  and in can once) .0; we have classified the variable into less than 2500 g, as low birth weight, or greater than 2500 g.Birth weight (in g) was reported by the mother at t1 with the question: has the mother or father of the study participant ever been diagnosed by a doctor as having hypertension or high blood pressure?Parental hypertension was collected at t0-percentiles was correlated with the rank order of t1-percentiles. For the last two groups, the rank order of t0-percentiles were correlated with the rank order of t1 mmHg values.The analysis was performed in two parts. First, BP tracking was estimated by calculating Spearman's rank-order correlation coefficients stratified by sex and age and potential hypertension risk factors . For this purpose, four age groups were chosen: 3 to 6, 7 to 10, 11 to 14, and 15 to 17 years. In the first two age groups, the rank order of thttp://comparingcorrelations.org/) [For testing statistical differences between correlation coefficients, we have used the software cocor (ns.org/) .0 and t1. For calculating the positive predictive value (PPV) of hypertensive BP at t0 for future hypertensive BP (six years later at t1), as well as the negative predictive value (NPV) of normotensive BP at t0 for future normotensive BP, we used the software MedCalc . We calculated the PPV and NPV also for risk factor combinations, e.g., for having hypertensive BP and being obese at t0. In sensitivity analyses, we checked whether the results were altered when using weighting factors in the analysis. These weights take selective participation in the MoMo study and the drop-out between t0 and t1 into account. They are the inverse of the probability to participate in the MoMo study/the follow-up, estimated in a logistic regression model including several relevant KiGGS variables as predictors. Since no weights are available for the exact population under study here, we used three existing sets of weights: longitudinal weights adjusting the KiGGS follow-up to the KiGGS baseline, longitudinal weights adjusting the MoMo follow-up to the MoMo baseline, and cross-sectional MoMo weights adjusting the MoMo follow-up to the KiGGS1 follow-up.In the second part of the analysis, we determined the proportion of participants with hypertensive BP measurement at t1-BP from t0-BP and other baseline and follow-up risk factors for hypertension, using logistic regression models. Models were run separately for the t0-age-group 3 to 10 years (percentile-defined t1-hypertension) and the t0-age-group 11 to 17 years (140/90 mmHg threshold t1-hypertension). STATA SE14.1 was used for the analyses.Furthermore, we investigated the prediction of hypertensive tThe characteristics of the 2542 study participants  at baseline (2003-2006) and six year follow-up (2009-2012) are presented in Spearman rank-order correlations for BP tracking from childhood to adolescence/young adulthood are shown in The BP tracking coefficients stratified by birthweight, parental hypertension and BMI trajectories remained in the order of magnitude of the tracking coefficients for the overall sex-age groups; i.e., children with these hypertension risk factors did not have substantially higher SBP or DBP tracking correlation coefficients compared to children without these risk factors (data not shown).1) and an older age group  (0 and t1) accounted for 3.1% of this age group; incident hypertensive BP  was observed in 11.6%; resolution of hypertensive BP  was observed in 4.8% and the proportion of persistently normal BP in the 3- to 10-year-old group accounted for 80.5%. Among children and adolescents aged 11 to 17 years, 11.2% (95% CI 8.6-14) had hypertensive BP at baseline. Of these, 34% (n = 20 out of 59) also had hypertensive BP at follow-up. In this age group, persistent hypertensive BP accounted for 3.8%; incident hypertensive BP was observed in 12%; resolution of hypertensive BP in 7.4% and persistently normal BP in 77% of the study population  . Among cpulation .1. In the models for both the younger and the older age group hypertensive BP at t0 was one of the major determinants (OR 4.25 (95% CI 2.90-6.23) and OR 3.35 (95% CI 1.60-7.01)). Further, increasing age was also identified as a risk factor for hypertensive BP at follow-up in both age groups. In the 3- to 10-year-old group, children with a persistently overweight/obesity as well as those with an incident overweight/obesity were more likely to have hypertensive BP at t1 (OR 2.70 (95% CI 1.78-4.12) and OR 4.44 (95% CI 3.08-6.42), respectively) compared to children who had a persistently low or normal BMI. A gender difference was found only in the 11- to 17-year-old group, in which females were less likely to have hypertensive BP at t1 compared with their male peers. Moreover, only in the 11- to 17-year-old group, parental hypertension was found to be a determinant for hypertensive BP at follow-up.The univariate and multivariable logistic regression  showed t0) with a probability of 88% (95% CI 47-99.7) of having hypertensive BP at t1.The probability of having hypertensive BP at six year follow-up increased with baseline BP percentile  but did The positive (PPV) and negative predictive values (NPV) according to age at baseline and risk factors are presented in This study investigates different aspects of BP tracking from childhood to adolescence/young adulthood with recent population-based data from Germany. Applying various metrics for assessing tracking including correlation coefficients, positive predictive values, and ORs from multivariable predictive modelling, BP tracking from childhood to adolescence and young adulthood was generally moderate. Our results confirm previous findings that BP in childhood and adolescence is one of the strongest predictors of future BP; however, it is one of the strongest predictors among several moderate or weak predictors. Among children and adolescents aged 3 to 17 years with hypertensive BP at baseline (mean of two measurements taken on one occasion), about one in three had hypertensive BP six years later. In population subgroups of children with additional hypertension risk factors such as overweight or parental hypertension, the PPV of baseline hypertensive BP increased but was not much higher than 50%.In line with previous findings, we found moderate BP tracking coefficients, higher for SBP than for DBP –3. StratOne could ask whether BP tracking coefficients become larger among children with additional risk factors for hypertension, but in fact mathematically they are expected to remain similar or even become smaller, as verified with data from this study (results not shown). This happens since in subgroups with additional hypertension risk factors, BP values are more similar than in an unselected group and ranking to a greater extent due to chance. In other words, for combinations of risk factors, tracking coefficients are not an appropriate measure for assessing the predictability of future BP values.Our results confirm previous findings that risk factors for hypertensive BP in young adulthood can be identified in childhood and adolescence , 17. HowThe association between overweight/obesity and hypertension in children was observed in several studies –20. Our Our univariable logistic regression analysis showed that children with hypertensive parents were at higher risk of hypertension at follow-up compared to children without hypertensive parents. However, this was only confirmed in the multivariable analysis of 11-17-year olds. Children and adolescents with parental hypertension had an 18% and 21% probability of having a hypertensive BP at follow-up . A famil0 was a significantly stronger predictor for hypertension at t1 than parental hypertension in both age groups , the six-year follow-up, and standardized BP measurements according to the same protocol at baseline and follow-up examinations. However, despite using the same BP measurement devices, identical resting times, and the same measurement protocol, including performing BP measurements before motor performance testing, we cannot exclude the possibility that the environment and expectation of an extensive motor testing like in the MoMo study could have influenced BP at follow-up, leading to somewhat higher BP values and somewhat lower tracking indices. Another limitation is the possible selection bias arising from selective participation in the MoMo study and from longitudinal drop-out. Sensitivity analyses with weights that partially account for this selection showed somewhat lower predictive values and thus did not alter the main conclusions. A further limitation is that the rule for KiGGS0 (2003-2006) was that the cuff should cover at least two-thirds of the upper arm length while current recommendations (2016 ESH guidelines ) suggestIn conclusion, our analysis with recent population-based data from Germany shows that BP in children and adolescents tracks only moderately over six years; however, BP in childhood is the strongest independent predictor for future hypertensive BP measurements.


Tribolium castaneum, using the area preference method, after four hours of exposure and the effect transcriptional of genes associated with neurotransmission. Changes in gene expression of acetylcholinesterase (Ace1), GABA-gated anion channel splice variant 3a6a (Rdl), GABA-gated ion channel (Grd), glutamate-gated chloride channel (Glucl), and histamine-gated chloride channel 2 (Hiscl2) were assessed and the interaction with proteins important for the insect using in silico methods was also studied. For linalool and β-pinene, the repellent concentration 50 (RC50) values were 0.11 µL/cm2 and 0.03 µL/cm2, respectively. Both compounds induced overexpression of Hiscl2 gen in adult insects, and β-pinene also promoted the overexpression of Grd and the Ace1 gene. However, β-pinene and linalool had little potential to dock on computer-generated models for GABA-gated ion channel LCCH3, nicotinic acetylcholine receptor subunits alpha1 and alpha2, and putative octopamine/tyramine receptor proteins from T. castaneum as their respective binding affinities were marginal, and therefore the repellent action probably involved mechanisms other than direct interaction with these targets. Results indicated that β-pinene was more potent than linalool in inducing insect repellency, and also had a greater capacity to generate changes in the expression of genes involved in neuronal transmission.Effective, ethical pest control requires the use of chemicals that are highly specific, safe, and ecofriendly. Linalool and β-pinene occur naturally as major constituents of the essential oils of many plant species distributed throughout the world, and thus meet these requirements. These monoterpenes were tested as repellents against  Biopesticides are gaining increased attention and interest among those concerned with environmentally friendly, safe, and integrated crop management approaches ,2 and, cTerpenes are a class of plant secondary metabolites that have a distinct structure and function, and are considered to be important agents in the medicinal uses of aromatic plants . MonoterT. castaneum, and assesses the interaction with proteins important for the insect using in silico methods.Linalool and β-pinene are two alcohol and hydrocarbon volatile monoterpenes  reported50) values of 0.11 and 0.03 µL/cm2 for linalool and β-pinene, respectively. The RC50 of the positive control was 0.02 µL/cm2. The two-way ANOVA results showed that there was interaction between the different concentrations evaluated, therefore, the repellent effect was dependent on the concentration.The percentage repellency results from the chemicals tested are presented in T. castaneum adults are presented in In this study, changes in the gene expression of The best proteins obtained in the homology modeling process were loaded in Protein Model Data Base (PMDB). The validation results obtained using the different servers demonstrated the quality of the models. ProQ rated the obtained models as fairly, very, and extremely good .The results of the validation process obtained by RAMPAGE showed that the studied proteins had a number of residues in favored and allowed regions of over 90% . FiguresT. castaneum proteins involved in neurological responses. The results are presented in A computational approach was carried out to identify possible targets for linalool and β-pinene, with a particular focus on the The 3D structure of the proteins and amino acids involved in the protein-ligand interaction are shown in 50 values than IR3535, although these were comparable with the positive control.Natural insect repellent products have become a viable alternative to synthetic repellents ,13 as thT. castaneum than linalool. This compound is an oxygenated monoterpene which is abundantly present in the environment, and is widely found in plants as a constituent of EOs [T. castaneum after three days of exposure [Aedes aegypti) [T. castaneum and Dominica Rhyzopertha) [β-pinene was more potent against t of EOs . It has exposure . Meanwhiexposure  that hasaegypti)  and beetopertha) , which cLinalool and β-pinene are insect repellents; however, the mechanisms by which these compounds function are not fully understood. It is only known that monoterpenes can act on various insect and mammalian receptors, especially at the level of the nervous system, and specifically on gamma-aminobutyric acid (GABA)-gated chloride channels; octopamine, tyramine, and nicotinic acetylcholine (nAChR) receptors; acetylcholine esterase; sodium channels; and other targets. Several different monoterpenoids have been reported to bind to ionotropic GABA receptors in humans, rodents, and insects in previous research ,5,19. AlDrosophila as well as in lobster stomatogastric, cardiac, and olfactory neurons [Repellent activity is related to changes in the behavior of insects, which are caused by effects on the CNS. The histamine-gated ion channel is a member of the cys-loop ligand-gated ion channel (cys-loop LGIC) superfamily , and has neurons ,24. AdulMonoterpenes are capable of quickly penetrating insect cells as they are volatile and lipophilic compounds that interfere with physiological functions so their modes of action or mechanisms of resistance are especially complex ,5. For iT. castaneum adults (T. castaneum function as GABA-gated chloride channels (GABACls-Grd), receptor members of the family of the Cys-loop ligand-gated ion channels, which mediate fast inhibitory synaptic transmission [Compared to β-pinene, linalool exhibited less activity in terms of gene expression, despite some potential to interact with proteins involved in synapsis . On the m adults . The ionsmission ,27. On tsmission . AlthougZ-score uses structures that are solved by X-ray crystallography as a reference to estimate the absolute quality of a model [Z-score was in the range of −10.99 to −4.70, showing that these models varied in quality; conversely, however, the results obtained with RAMPAGE and ProQ suggested that the models had the appropriate quality [The use of molecular tools together with in silico approaches assists in gaining insight into toxicity mechanisms ,30,31. I a model . For the quality ,35,36.T. castaneum proteins involved in the neurotransmission process  and β-pinene (>95%) were purchased from Sigma-Aldrich . Acetone reagent (≥99.5%) was also purchased from Sigma-Aldrich.T. castaneum under laboratory conditions. The repellent activity of linalool and β-pinene was evaluated using the area preference method [2). As experimental containers, Petri dishes (9 cm in diameter) were used to house 20 T. castaneum. Linalool and β-pinene were serially dissolved in acetone . Five hundred µL of the solution were uniformly applied to a half-filter paper disc to five different testing concentrations. The other half of the remaining filter paper was treated with 500 µL of acetone and used as the control vehicle. Then, the half discs were air-dried for about 10 min to allow solvent evaporation. During each test, 20 T. castaneum adults without regard to gender were released at the center of the disk, then covered quickly with a dish cover and placed in darkness. Counts of the insects present on each side of the filter paper were made after 4 h of exposure. As a positive control, a 15% formulation of IR3535 ethyl 3-(N-acetyl-N-butylamino)propionate [The experiments were carried out on adults of e method ,40. Test50) for each compound was obtained by Probit analysis  and was used for gene expression assays.After the experiment was conducted, the median repellent concentration  according to the manufacturer’s protocols. A real-time polymerase chain reaction (RT-PCR) was performed on a StepOne Plus real-time PCR system  apparatus. Six genes were analyzed  according to the manufacturer’s protocols. RNA concentrations were measured using a NanoDrop 2000 spectrophotometer  and the quality was verified by absorbance ratio 260/280 nm. cDNA was obtained through the QuantiTectanalyzed  [41,42,4T. castaneum were obtained from Uniprot [The amino acid sequences of the proteins from  Uniprot  in FASTA Uniprot ,50, Phyr Uniprot , and I-T Uniprot . The bes Uniprot . The qua Uniprot , ProQ . MGLTools 1.5.0 software  was utilized to convert structures from PDB to PDBQT format, adding polar hydrogens and assigning Kollman partial charges. The 3D structure of the linalool and β-pinene were obtained from Pubchem [Prior to molecular docking, the 3D structures of carboxylic ester hydrolase, carboxylic ester hydrolase 2, GABA-RDL, GABA-GRD, GABA-gated ion channel LCCH3, glutamate-gated chloride channel, histamine-gated chloride channel 1, histamine-gated chloride channel 2, hormone receptor in 39-like, nicotinic acetylcholine receptor subunit alpha1, nicotinic acetylcholine receptor subunit alpha2, pH sensitive chloride channel, and putative octopamine/tyramine receptor proteins from  Pubchem  and opti Pubchem . Docking Pubchem . Docking Pubchem . The exi Pubchem . The val Pubchem .t-test was utilized to compare the mean number of insects on the treated and untreated areas of the filter paper. Repellency or attraction was established if significant differences occurred for positive or negative percentage repellency, respectively. Probit analysis was employed to calculate RC50. Normal distribution and equality between variances were checked by the Kolmogorov–Smirnov and Bartlett’s tests, respectively. Two-way analysis of variance (ANOVA) with the Bonferroni post-test was used to compare all groups (treated and control) at different time intervals. The data are presented as means ± SE and the differences between means are considered to be significant at p ≤ 0.05. GraphPad InStat 3.05  was used for data analysis. The parameters evaluated in the statistical analysis are found in The paired T. castaneum, affecting the expression of genes related to neurotransmission such as acetylcholinesterase, GABA-gated ion channel, and histamine-gated chloride channel 2. However, each chemical compound induced a different gene expression profile. These chemicals had little ability to dock on proteins associated with the neurotransmission process in the red flour beetle.In summary, the monoterpenes linalool and β-pinene showed repellent activity against